Navigation Links
Transformation of Nematodes With the Helios Gene Gun

Peter Jackstadt1, Horst Zahner2 and Gerd Hobom1, 1 Institut fr Mikrobiologie und Molekularbiologie der Justus-Liebig Universitt Gieen, Frankfurter Str. 107, D-35392, Gieen, 2 Institut fr Parasitologie der Justus-Liebig-Universitt Gieen, Rudolf-Buchheim-Str. 2, D-35392 Gieen


Introduction
The Biolistic DNA transfer technique was first established for plant cells and plant tissues (Sandford et al., 1987; Jefferson et al., 1987; Bruce et al., 1989; Armaleo et al., 1990). More recent applications have been DNA transfer experiments aiming at DNA vaccination of animals (genetic immunization; Sundaram et al., 1996), for example a bombardment of mice to generate protective antibodies, instead of the DNA injection used for this purpose in the past.

Here we report on the potential to use this convenient method for transfection of nematodes, not only for the free-living soil nematode Caenorhabditis elegans, the well known model organism in its class, but also for the cotton rat filaria Litomosoides sigmodontis, which serves as model for human pathogenic filariae (Zahner, Hobom and Stirm, 1995), such as Wuchereria bancrofti or Brugia spp. These parasitic species cause a severe lymphatic disease called filariasis (elephantiasis), from which more than 100 million human beings suffer worldwide.


Methods
PREPARATION OF ADULT HERMAPHRODITE C. ELEGANS AND COTTON RAT PARASITE L. SIGMODONTIS FOR MICROPARTICLE BOMBARDMENT
C. elegans is cultivated on NGM agar plates (Brenner, 1974). Wild-type worms (strain N2 var. Bristol) are fed on Escherichia coli OP-50 (exogenous uracil dependent mutant) seeded on those plates. The surface of a densely populated NGM agar plate is subsequently rinsed with tap water. A drop of water containing nematodes is placed in the center of a 6 cm Petri dish on ice (to reduce their motility). In this state C. elegans could be used for microparticle bombardment (Figure 1A).

For the same purpose adult females of L. sigmodontis must be isolated from the pleural cavity of their laboratory host Mastomys coucha (Wegerhof and Zahner, 1985), and stored in RPMI medium (supplemented with 10% FCS, Gibco-BRL; 1% Penicillin and Streptomycin). Single parasites are transferred to an agar plate and used for microprojectile bombardment.

CONDITIONS OF BOMBARDMENT
DNA-coated gold particles are chosen as microparticles following a modified Bio-Rad protocol for the Helios gene gun. The protocol is as follows:
1. Mix 50 mg of gold microparticles (Bio-Rad; a 1:1 mixture of particles 0.6 /1.0 in diameter) with 100 l of 0.05 M spermidine (Sigma), and sonicate 3 times for 3 seconds in an Eppendorf tube (1.5 ml).

2. Add 200 l of purified supercoiled plasmid DNA to the carrier suspension (in a total volume of 100 l). It is possible to use more than one plasmid simultaneously.

3. CaCl2 (100 l, 1 M) is now added dropwise with continuous vortexing. Precipitation was allowed to take place for 10 minutes at room temperature.

4. Wash the suspension three times with 1 ml of absolute ethanol (Sigma-Aldrich), and pellet the sample by centrifugation (12,000 rpm for 1 minute, Heraeus Biofuge).

5. Finally, the pellet is resuspended in 3.5 ml of ethanol containing 0.1 mg/ml polyvinylpyrrolidone (PVP; MW 360,000). In this state a DNA-coated gold particle suspension can be stored for up to 2 months at -20 C (if not directly used for cartridge preparation).

USING THE TUBING PREP STATION
1. Clean the inner surface of the Tefzel tubing (external size 10 mm; internal diameter 6 mm; Bio-Rad) with 5 ml of absolute ethanol and dry with nitrogen (grade 4.8; 99.998% pure) in the rotating tubing holder for at least 5 minutes.

2. Sonicate the DNA-coated particle suspension in ethanol for 3 seconds (to achieve a homogeneous solution) and load it into the tubing at a rate of 5.56 ml/min using a syringe.

3. Allow the DNA-coated particles to settle for 3 minutes.

4. Remove the ethanol solution quickly (within 30 seconds).

5. For drying the wall-attached particles, flush nitrogen gas through the tubing for 3 minutes at a flow rate of 0.35 l/min.

6. Finally, cut the resulting particle-covered tubing into individual cartridges 16 mm in length (one full length of tubing results in nearly 50 cartridges) to fit into the 12-shot barrels of the Helios gene gun (Bio-Rad). See Figure 2.

7. In a desiccated atmosphere, these can be stored at 4 C for up to 8 months without losing activity.

8. Deliver the DNA-coated gold particles using 4 blasts of helium (grade 4.5; 99.995% pure) applied to the cartridges at a pressure of 300 p.s.i. and a distance of 3 cm to the target (in the case of C. elegans).

9. A pressure of 120180 p.s.i. at a distance of 2 cm using 1 blast of helium is useful for the transfer into parasitic nematode L. sigmodontis. After this procedure, the female parasites may be cultivated at 37 C for up to 2 weeks in RPMI medium (5% CO2).

SELECTION FOR TRANSFORMED TRAITS OF C. ELEGANS
While the majority (up to 90%) of C. elegans hermaphrodites are killed during Biolistic DNA transfer, the survivors with their gold particles incorporated may be cultivated on new E. coli-seeded NGM agar plates (Figure 2A shows particles within a living hermaphrodite). F1/F2 progeny nematodes were screened 3 to 4 days later for visible dominant mutant marker rol-6 (Kramer et al., 1990 ). Animals carrying roller allele su-1006 exhibit a helically twisted cuticle and body. Muscle contractions, which would normally propel the worm forward or backward in a sweeping sinusoidal motion, will cause the nematode instead to roll over its longitudinal axis and to move in circles (Mello et al., 1991; Fire, 1986; Kramer et al., 1990). In the case of L. sigmodontis, no special selection mode is necessary because the success of bombardment can be visualized by standard light microscopy to make visible the gold particles now located within these filariae (see Figure 2B).


Results
The established mode of DNA transfer into C. elegans is microinjection of DNA into the gonadal syncytium (Stinchcomb et al., 1985; Fire, 1986). Under standard selection conditions promoter-reporter gene cDNA constructs are used, in co-transfection with a visible dominant such as mutation rol-6 (su1006), which leads to roller movement phenotype of transformed worms (already in F1 generation) and is easily detectable within a background of normally moving animals. The same transfection efficiency has been observed for this Biolistic DNA transfer method as for microinjection experiments (Fire, 1986), while this new strategy is less time consuming and much more convenient for co-transfection experiments. In the F1 generation, DNA transfer rates of up to 1% were observed using this selection method, which became considerably enriched during following generations after further transfer (F3/F4: 20%; F5/F6 up to 90%), proving their genetic fixation.

Besides marker gene rol-6, we have employed two different reporter genes, a mutant form of GFP (green fluorescent protein), which was originally cloned from the jellyfish Aequorea victoria (Prasher et al., 1992; Cody et al., 1993; Chalfie et al., 1994) with enhanced fluorescence (Siemering et al., 1996), and -galactosidase from Escherichia coli. Expression was visualized by standard fluorescence microscopy (GFP; see Figure 4) or by histochemical staining (lacZ).

In both cases we have observed marker gene expression under transcriptional control of the SV40 early promoter (Figure 3 A and B), or of the C. elegans actin-1 core promoter after Helios gene gun mediated delivery of Au/DNA particles into the nematode. The lacZ expression of constructs containing the actin-1 core promoter of C. elegans (Files et al., 1983; Krause et al., 1989; Stone and Shaw, 1993) in DNA transfected L. sigmodontis will even be analyzed histochemically.


Discussion
The Biolistic DNA-transfection procedure, because of its simple mode of handling, has an important potential in various innovative molecular biology applications, such as DNA vaccination, protein targeting, and basic research applications such as gene regulation studies. The new Biolistic technology should therefore be able to substitute for other applications in this field. Also, the prepared DNA/Au particles can be stored for rather long periods, at least up to 8 months, which makes repetitions or comparative experiments more meaningful. In applying this technique to various nematodes, it is conveniently possible to use a couple of hundred target worms of C. elegans and proceed in comparatively low precision, while DNA injections have to be done individually and precisely into the gonads. Parasitic worms, however, can only be transfected on an individual basis or in small groups by Biolistic gene transfer. In all experiments designed, reporter gene expression is observed independent from distance of initial gold particle target sites, clearly indicating an DNA drift within the entire bodies. Altogether, this technique is a reproducible and efficient method of producing fertile transgenic nematodes.


References
1. Armaleo, D., Ye, G.-N., Klein, T. M., Shark, K. B., Sandford, J. C. and Johnston, S. A., Curr. Genet., 17, 97103 (1990).

2. Brenner, S., Genetics, 77, 7194 (1974).

3. Bruce, W. B., Christensen, A. H., Klein, T., Fromm, M. and Quail, P. H., PNAS, 86, 96929696 (1989).

4. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C., Science, 263, 802804 (1994).

5. Cody C. W., Prasher, D. C., Westler, W. M., Prendergast, F. G. and Ward, W. W., Biochemistry, 32, 12121218 (1992).

6. Files, J. G., Carr, S. and Hirsh, D., J. Mol. Biol., 164, 355375 (1983).

7. Fire, A., EMBO J., 5, 26732680 (1986).

8. Franz, M. and Andrews, P., Z. Parasitenkd., 72, 387395 (1986).

9. Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W., EMBO J., 6, 39013907 (1987).

10. Kramer, J. M., French, R. P., Park, E. and Johnson, J. J., Mol. Cell Biol., 10, 20812090 (1990).

11. Krause, M., Wild, M., Rosenzweig, B. and Hirsh, D., J. Mol. Biol., 208, 381392 (1989).

12. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. and Cormier, M. J., Gene, 111, 229233 (1992).

13. Sandford, J. C., Klein, T. M., Wolf, E. D. and Allen, N., Part. Sci. Technol., 5, 2737 (1987).

14. Siemering, K. R., Golbik, R., Sever, R. and Haseloff, J., Current Biology, 6, 16531663 (1996).

15. Stinchcomb, D., Shaw, J. E., Carr, S. H. and Hirsh, D., Mol. Cell Biol., 5, 34843496 (1985).

16. Stones, S. and Shaw, J. E., Gene, 131, 167173 (1993).

17. Sundaram, P., Xiao, W. and Brandsma, J. L., Nucleic Acids Research, 7, 13751377 (1996).

18. Zahner, H. and Wegerhof, P. H., Z. Parasitenkd., 71, 583593 (1985).

19. Zahner, H., Hobom, G. and Stirm, S., Parasitology Today (Review), 11, 116120 (1995).


Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) via Sonderforschungsbereich 535. We thank Noelia Pohl, Ulrike Ruppert and Brigitte Hofmann for their exceptional technical assistance. Many thanks to PD Dr. R. Dennis for helpful discussion and comments on the manuscript.


Biolistic is a registered trademark of E. I. duPont de Nemours and Co.
Biolistic technology is exclusively licensed to Bio-Rad Laboratories.
Eppendorf is a trademark of Eppendorf Geratebau Netheler + Hinz, GmbH.


back to top
'"/>

Source:


Page: All 1 2 3 4 5 6 7

Related biology technology :

1. Highest Possible Transformation Efficiencies for High-Throughput Applications
2. New Competent Cells for Highest Transformation Efficiencies
3. Optimization of Biolistic Transformation Using the Helium-Driven PDS-1000/He System
4. Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cells
5. Transformation of Filamentous Fungi by Microprojectile Bombardment
6. Transformation of Filamentous Fungi by High-Voltage Electroporation
7. Delivery of pCMV-S DNA Using the Helios Gene Gun System Is Superior to Intramuscular Injection in Balb/c Mice
8. Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the Helios Gene Gun
9. Detection of Reporter Gene Activity in Cell Cultures and Murine Epidermis After Helios Gene Gun-Mediated Particle Bombardment
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Transformation Nematodes With the Helios Gene Gun

(Date:10/30/2014)... Spartan Bioscience announced today that Health Canada has ... detects CYP2C19 genetic mutations in less than 60 minutes ... CYP2C19 System is the first near-patient DNA test for ... to the system’s ease of use, Health Canada has ... such as doctors, nurses, pharmacists, and laboratory technicians, in ...
(Date:10/30/2014)... Calif. , Oct. 30, 2014 Isis Pharmaceuticals, ... therapeutics, today announced that management will present a company overview ... at 8:30 a.m. ET in Boston, MA. ... be available on the "Investors & Media" section of the ... on the Isis website within 48 hours and will be ...
(Date:10/30/2014)... (Oct. 29, 2014) – The severe flooding that devastated ... been less destructive if the bridges, roads and other ... new study from the University of Colorado Denver. , ... infrastructure," said Jimmy Kim, PhD, associate professor of structural ... Applied Science and lead author the study. "There is ...
(Date:10/27/2014)... and TORONTO , Oct. 27, ... ) (OTCQB: GNBT) today announced two presentations demonstrating ... by its novel proprietary cancer immunotherapeutic AE37 vaccine ... cancer patients. The AE37 cancer vaccine is being ... Inc. ( www.antigenexpress.com ). The presentations are being ...
Breaking Biology Technology:Health Canada approves first near-patient DNA test for personalized medicine 2Health Canada approves first near-patient DNA test for personalized medicine 3Isis Pharmaceuticals to Present at the Nomura Biotechnology Conference 2CU Denver study says upgrading infrastructure could reduce flood damage 2Generex Announces Presentations Correlating Immunological Response with Reduced Recurrence in Phase II Study of AE37 Breast Cancer Vaccine in Patients at the American College of Surgeons 2Generex Announces Presentations Correlating Immunological Response with Reduced Recurrence in Phase II Study of AE37 Breast Cancer Vaccine in Patients at the American College of Surgeons 3Generex Announces Presentations Correlating Immunological Response with Reduced Recurrence in Phase II Study of AE37 Breast Cancer Vaccine in Patients at the American College of Surgeons 4
... , WILMINGTON, Delaware, November ... Cell and Advanced Technologies Market, (2009 - 2014),published ... in the global stem cell market. It identifies ... for the various,products, services, technologies, and applications submarkets ...
... -- (Nov. 2, 2009) -- Rice University scientists today ... carbon-nanotube fibers that could lead to revolutionary advances in ... a nine-year program, the method builds upon tried-and-true processes ... plastics. The research is available online in the journal ...
... Nov. 2 BioBehavioral Diagnostics Company announced today that ... it was accepted via a rigorous peer-review process and ... Annual Meeting of the American Academy of Child and ... 2009. Calvin R. Sumner, M.D., the company,s Chief Medical ...
Cached Biology Technology:MarketsandMarkets: Global Stem Cell and Advanced Technologies Market Worth US$ 88.3 Billion By 2014 2MarketsandMarkets: Global Stem Cell and Advanced Technologies Market Worth US$ 88.3 Billion By 2014 3Breakthrough in industrial-scale nanotube processing 2Breakthrough in industrial-scale nanotube processing 3Breakthrough in industrial-scale nanotube processing 4Objective Measures of ADHD Symptoms Using the Quotient(TM) ADHD System May Reduce Cost of ADHD Drug Trials 2Objective Measures of ADHD Symptoms Using the Quotient(TM) ADHD System May Reduce Cost of ADHD Drug Trials 3Objective Measures of ADHD Symptoms Using the Quotient(TM) ADHD System May Reduce Cost of ADHD Drug Trials 4
(Date:10/29/2014)... Mich. – As bodies decompose, their types and ... clues they provide could mean the difference between ... Michigan State University is using a more than ... Detroit death-scene investigators examine these changing populations. The ... geographical location of death, gender, race, socioeconomic relations ...
(Date:10/29/2014)... in the living world compete with members of ... how much they invest into their competitive ability. ... get access to high-quality resources, while others seem ... the lower-quality resources that are left over for ... and human societies seems to fluctuate considerably over ...
(Date:10/28/2014)... release is available in German . ... relatives, the chimpanzees, acquire sufficient food when times are ... Park in Côte d,Ivoire researchers of the Max Planck ... clear example of how great apes can acquire extra ... that chimpanzees make their sleeping nests more en route ...
Breaking Biology News(10 mins):MSU partners with Detroit to investigate death scenes 2Evolution of competitiveness 2Evolution of competitiveness 3The early chimp gets the fig 2The early chimp gets the fig 3
... Wildfire, insect outbreaks and hurricanes destroy huge amounts of ... dioxide entering the atmosphere, but scientists are now learning ... climate impact. Researchers conclude in a new study ... energy reflected back into space, is important in the ...
... river to connect two major metropolises, the corpus callosum ... left and right hemispheres of our brains. Now, neuroscientists ... that people who are born without that linka condition ... remarkably normal communication across the gap between the two ...
... represent the most richly diverse class of creatures in the ... serves as a review of the 27 orders of British ... overall biology and importance of more than 500 families. ... all the British insect groups which can appeal to the ...
Cached Biology News:'Albedo effect' in forest disturbances can cause added warming, bonus cooling 2'Albedo effect' in forest disturbances can cause added warming, bonus cooling 3Bridging the gap 2Bridging the gap 3
... Clarin represents the state-of-the-art in ... for the permanent preservation of immunohistochemical ... (DAB), Vega Red, New Fuchsin, and ... substrates on tissue sections. Clarin can ...
... Gene Inspector can perform many sophisticated DNA analysis ... much more than just another sequence analysis product. ... suite of analyses to be performed each time ... custom style sheets which specify exactly how your ...
... columns is a well-known separation technique in ... are in DNA/RNA extraction in microbiology, viral ... in chemical or analytical laboratories. ,This ... series instruments for smaller scales. The extraction ...
... is a revolutionary new application for ... a novel interface, Expression makes complex ... simple. Expression uses the very latest ... in the way sequences are analysed. ...
Biology Products: